METHOD TO-5
Revision 1.0
April, 198 4
METHOD FOR THE DETERMINATION OF ALDEHYDES AND KETONES IN AMBIENT
AIR USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
1. Scope
1.1 This document describes a method for determination
of individual aldehydes and ketones in ambient air.
With careful attention to reagent purity and other
factors the method can detect most monofunctional
aldehydes and ketones at the 1-2 ppbv level.
1.2 Specific compounds for which the method has been
employed are listed in Table 1. Several studies
have used the same basic method, with minor
procedural differences, for analysis of ambient air
(1-3) .
2. Applicable Documents
2.1 ASTM Standards:
D 1356 Definitions of Terms Related to Atmospheric
Sampling and Analysis(s)
2.2 Other Documents
Ambient air studies (1-3)
U.S. EPA Technical Assistance Document (4)
3. Summary of Method
3.1 Ambient air is drawn through a midget impinger
containing 10 mL of 2N HCl/0.05% 2,4-
dinitrophenylhydrazine (DNPH reagent) and 10 mL of
isooctane. Aldehydes and ketones readily form
stable 2,4-dinitrophenylhydrazones (DNPH
derivatives).
3.2 The impinger solution is placed in a screw-capped
vial having a teflon-lined cap and returned to the
laboratory for analysis. The DNPH derivatives are
recovered by removing the isooctane layer,
extracting the aqueous layer with 10 mL of 70/30
hexane/methylene chloride, and combining the
organic layers.
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3 . 3
The combined organic layers are evaporated to
dryness under a stream of nitrogen and the residue
dissolved in methanol.
3.4 The DNPH derivatives are determined using reversed
phase HPLC with an ultraviolet (UV) adsorption
detector operated at 370 nm.
Significance
4.1 Aldehydes and ketones are emitted into the
atmosphere from chemical operations and various
combustion sources. In addition, several of these
compounds (e.g., formaldehyde and acetaldehyde) are
produced by photochemical degradation of other
organic compounds. Many of these compounds are
acutely toxic and/or carcinogenic, thus requiring
their determination in ambient air in order to
assess human health impacts.
4.2 Conventional methods for aldehydes and ketones have
generally employed colorimetric techniques wherein
only one or two compounds are detected, or the sum
of numerous compounds is determined. The method
described herein provides a means for specifically
determining a wide variety of aldehydes and ketones
at typical ambient concentrations.
Definitions
Definitions used in this document and any user prepared SOPs
should be consistent with ASTM D1356(5). All abbreviations
and symbols are defined within this document at the point of
use.
Interferences
6.1 The only significant interferences in the method
are certain isomeric aldehydes or ketones which may
be unresolved by the HPLC system. Such
interferences can often by overcome by altering the
separation conditions (e.g., using alternate HPLC
columns or mobile phase compositions).
6.2 Formaldehyde contamination of the DNPH reagent is a
frequently encountered problem. The reagent must
be prepared within 48 hours before use and must be
stored in an uncontaminated environment before and
after sampling to minimize blank problems. Acetone
contamination is apparently unavoidable.
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Consequently, the method cannot be used to
accurately measure acetone levels except in highly
contaminated environments.
7. Apparatus
7 . 1
7.2
7.3
7.4
7.5
7 . 6
7.7
7 . 8
7 . 9
7 .10
7 .11
7 . 12
7 . 13
7 . 14
7 . 15
7.16
Isocratic HPLC system-consisting of high pressure
pump, injection valve, Zorbax ODS column (25 cm x
4 . 6 mm ID) , variable wavelength UV detector, and
data system or stripchart recorded. See Figure 3.
Sampling system-capable of accurately and precisely
sampling 100-1000 mL/minute of ambient air. See
Figure 1.
Stopwatch
Friction top metal can, e.g., one-gallon (paint
can) - to hold DNPH reagent and samples
Thermometer - to record ambient temperature
Barometer (optional)
Analytical balance - 0.1 mg sensitivity
Reciprocating shaker
Midget impingers - jet inlet type - 25 mL volume
Ice bath - for cooling impingers during sampling
Nitrogen evaporator with heating block - for
concentrating samples
Suction filtration apparatus - for filtering HPLC
mobile phase.
Volumetric flasks - 100 mL and 500 mL.
Pipettes - various sizes, 1-10 mL.
Helium purge line (optional) - for degassing HPLC
mobile phase.
Erlenmeyer flask, 1-liter - for preparing HPLC
mobile phase.
7 .17
Graduated cylinder, 1 liter - for preparing HPLC
mobile phase.
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7.18 Microliter syringe, 10-25 jj,L - for HPLC injector.
8. Reagents and Materials
8.1 Bottles, 10 oz. glass, with teflon-lined screw cap
- for storing DNPH reagent.
8.2 Vials, 50 mL, with teflon-lined screw cap - for
holding samples and extracts.
8.3 Disposable pipettes and bulbs.
8.4 Granular charcoal.
8.5 Methanol, hexane, methylene chloride, isooctane -
distilled in glass or pesticide grade.
8.6 2, 4-Dinitrophenylhydrazine - highest purity
available (20% moisture).
8.7 Nitrogen, compressed gas cylinder - 99.99% purity
for sample evaporation.
8.8 Polyester filters, 0.22 //m - Nuclepore or equiv.
8.9 DNPH derivatives of the components of interest -
synthesized from DNPH and neat aldehydes according
to reference (7). Recrystalized from ethanol
before use.
9. Preparation of DNPH Reagent
9.1 Each batch of DNPH reagent should be prepared and
purified within 48 hours of sampling, according to
the procedure described in this section.
9.2 Two hundred and fifty milligrams of solid 2,4-
dinitrophenylhydrazine and 90 mL of concentrated
hydrochloric acid are placed into a 500 mL
volumetric flask and the flask is filled to the
mark with reagent water. The flask is then
inverted several times or sonified until all of the
solid material has dissolved.
9.3 Approximately 400 mL of the DNPH reagent is placed
in a 16 ounce glass screw-capped bottle having a
teflon-lined cap. Approximately 50 mL of a 70/30
(V/V) hexane/methylene chloride mixture is added to
the bottle and the capped bottle is shaken for 15
minutes on a reciprocating shaker. The organic
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layer is then removed and discarded by decanting as
much as possible and using a disposable pipette to
remove the remaining organic layer.
9.4 The DNPH reagent is extracted two more times as
described in 9.3. The bottle is then tightly
capped, sealed with teflon tape, and placed in a
friction top can (paint can) containing a 1-2 inch
layer of granulated charcoal. The bottle is kept
in the sealed can prior to use.
9.5 A portion of the DNPH reagent is analyzed using the
procedure described in Section 11 prior to use in
order to ensure that adequate background levels are
maintained.
10. Sampling
10.1 The sampling apparatus is assembled and should be
similar to that shown in Figure 1. EPA Method 6
uses essentially the same sampling system (8) . All
glassware (e.g., impingers, sampling bottles, etc.)
must be thoroughly rinsed with methanol and oven
dried before use.
10.2 Prior to sample collection the entire assembly
(including empty sample impingers) is installed and
the flow rate checked at a value near the desired
rate. In general flow rates of 100-1000 mL/minute
are useful. Flow rates greater than -1000
mL/minute should not be used because impinger
collection efficiency may decrease. Generally
calibration is accomplished using a soap bubble
flow meter or calibrated wet test meter connected
to the flow exit, assuming the entire system is
sealed. ASTM Method D3686 describes an appropriate
calibration scheme not requiring a sealed flow
system downstream of the pump.
10.3 Ideally a dry gas meter is included in the system
to record total flow. If a dry gas meter is not
available the operator must measure and record the
sampling flow rate at the beginning and end of the
sampling period to determine sample volume. If the
sampling period exceeds two hours the flow rate
should be measured at intermediate points during
the sampling period. Ideally a rotameter should be
included to allow observation of the flow rate
without interruption of the sampling process.
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10.4 To collect an air sample two clean midget impingers
are loaded with 10 mL of purified DNPH reagent and
10 mL of isooctane. The impingers are connected in
series to the sampling system and sample flow is
started. The following parameters are recorded on
the data sheet (see Figure 3 for an example) :
date, sampling location, time, ambient temperature,
barometric pressure (if available), relative
humidity (if available), dry gas meter reading (if
appropriate), flow rate, rotameter setting, DNPH
reagent batch number, and dry gas meter and pump
identification numbers.
10.5 The sampler is allowed to operate for the desired
period, with periodic recording of the variables
listed above. The total flow should not exceed -80
liters. The operator must ensure that at least 2-3
mL of isooctane remains in the first impinger at
the end of the sampling interval (i.e., for high
ambient temperatures lower sampling volumes may be
required).
10.6 At the end of the sampling period the parameters
listed in 10.4 are recorded and the sample flow is
stopped. If a dry gas meter is not used the flow
rate must be checked at the end of the sampling
interval. If the flow rate at the beginning and
end of the sampling period differ by more than 15%
the sample should be marked as suspect.
10.7 Immediately after sampling the impingers are
removed from the sampling system. The contents of
the first impinger are emptied into a clean 50 mL
glass vial having a teflon-lined screw cap. The
first impinger is then rinsed with the contents of
the second (backup) impinger and the rinse solution
is added to the vial. The vial is then capped,
sealed with teflon tape and placed in a friction
top can containing 1-2 inches of granular charcoal.
The samples are stored in the can, refrigerated
until analysis.
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10.8 If a dry gas meter or equivalent total flow
indicator is not used the average sample flow rate
must be calculated according to the following
equation:
0A=
Q1+Q2
CN
N
where
Qa = Average flow rate in mL/minute.
Qlf Q2. . . . Qn = Flow rate determined at
the beginning, end, and
intermediate points
during sampling.
N = Number of points averaged.
10 . 9
The total flow is
following equation:
then calculated using the
V =¦
' T ~T ) Q
¦ 2 1 '
1000
Vm = Total volume sampled in liters at
measured temperature and pressure
T2 = Stop time
Tx = Start time (T2 - Tx given in minutes)
11. Sample Analysis
11.1 Sample Preparation
11.1.1 The samples are returned to the laboratory in
50 mL screw-capped glass vials. To recover the
DNPH derivatives the following procedure is
employed.
11.1.2 The vials are shaken in a horizontal position
on a reciprocating shaker for 10 minutes. The
vials are then removed from the shaker and the
isooctane layer is removed and placed in a
second clean 50 mL screw-capped glass vial
using a disposable pipette.
11.1.3 The remaining aqueous layer is extracted with
10 mL of 70/30 (V/V) hexane/methylene chloride
in the same manner as described in 11.1.2.
The organic layer is removed and combined with
the isooctane extract.
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11.1.4 The combined organic extracts are then
concentrated to dryness at 40°C under a stream
of pure nitrogen. When the sample just
reaches dryness the vial is removed from the
nitrogen stream and a measured volume (2-5 mL)
of methanol is added to the vial. The vial is
tightly capped and stored refrigerated until
analysis.
11.2 HPLC Analysis
11.2.1 The instrument is assembled and calibrated as
described in Section 12. Prior to each
analysis the detector baseline is checked to
ensure stable operation.
11.2.2 A 5-25 iiL aliquot of the sample, dissolved in
methanol, is drawn into a clean HPLC injection
syringe. The sample injection loop is loaded
and an injection is made. The data system, if
available, is activated simultaneously with
the injection and the point of injection is
marked on the stripchart recorder.
11.2.3 After approximately one minute, the injection
valve is returned to "load" position and the
syringe and valve are flushed with methanol in
preparation for the next sample analysis.
11.2.4 After elution of the last component of
interest the acquisition is terminated and the
component concentrations are calculated as
described in Section 13.
11.2.5 After a stable baseline is achieved the system
can be used for further sample analyses as
described above.
11.2.6 If the concentration of a component exceeds
the linear range of the instrument the sample
should be diluted with methanol, or a smaller
volume can be injected onto the HPLC.
HPLC Assembly and Calibration
12 . 1
The HPLC system is assembled as shown in Figure 3.
The typical chromatographic performance and
operating parameters are shown in Figure 4.
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12.2 Mobile phase is prepared by mixing 800 mL of
methanol and 200 mL of reagent water. This mixture
is filtered through a 0.22 //m polyester membrane
filter in all glass and teflon suction filtration
apparatus. The filtered mobile phase is degassed
by purging with helium gas for 10-15 minutes (-100
mL/minute) or by heating to ~60°C for 5-10 minutes
in an Erlenmeyer flask covered with a watch glass.
A constant back pressure restrictor (-50 psi) or
short length (6-12 inches) of 0.01 inch I.D. teflon
tubing should be placed after the detector to
further eliminate mobile phase outgassing.
12.3 The mobile phase is placed in the HPLC solvent
reservoir and the pump flow is set at 1 mL/minute
and allowed to pump for 20-30 minutes prior to the
first analysis. The detector is switched on at
least 30 minutes prior to the first analysis and
the detector output is displayed on a stripchart
recorder or similar output device at a sensitivity
of .008 absorbance units full scale (AUFS). Once a
stable baseline is achieved the system is ready for
calibration.
12.4 Calibration standards are prepared in methanol from
the solid DNPH derivatives. Individual stock
solutions of -100 mg/L are prepared by dissolving
10 mg of the solid derivative in 100 mL of
methanol. These individual solutions are used to
prepare calibration standards containing all of the
derivatives of interest at concentrations of 0.1 -
10 mg/L, which spans the concentration of interest
for most ambient air work.
12.5 All calibration runs are performed as described for
sample analyses in Section 11. Before initial use
the operator should inject a series of calibration
standards (at least three levels) spanning the
concentration range of interest. Using the UV
detector, a linear response range of approximately
0.1 to 10 mg/L should be achieved, for -10 //L
injection volumes. Linear response is indicated
where a correlation coefficient of a least 0.999
for a linear least squares fit of the data
(concentration versus area response) is obtained.
12.6 Once linear response has been documented an
intermediate concentration standard near the
anticipated levels for each component, but at least
10 times the detection limit, should be chosen for
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daily calibration. The response for the various
DNPH components should be within 10% day to day.
If greater variability is observed more frequent
calibration may be required to ensure that valid
results are obtained.
12.7 The response for each component in the daily
calibration standard is used to calculate a
response factor according to the following
equation:
where
RFC = response factor for the component of
interest in nanograms injected/response
unit (usually area counts).
Cc = concentration of component in the daily
calibration standard (mg/L).
Vx = volume of calibration standard injected
{liL) .
Rc = response for component of interest in
calibration standard (area counts).
13. Calculations
13.1 The volume of air sampled is often reported
uncorrected for atmospheric conditions (i.e. under
ambient conditions). However, the value can be
adjusted to standard conditions (25°C and 760 mm
pressure) using the following equation:
298
V =V x——x
760 213 + Ta
where
Vs = total sample volume at 25°C and 7 60 mm Hg
pressure (liters) .
Vm = total sample volume under ambient
conditions (liters). Calculated in 10.9
or from dry gas meter reading.
PA = ambient pressure (mmHg).
Ta = ambient temperature (°C) .
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13.2
The concentration of each aldehyde (as the DNPH
derivative) is calculated for each sample using the
following equation:
W =RF xR jc-
V
where
Wd = total quantity of derivative in the
sample (//g) .
RFC = response factor calculated in 12.7
Rd = response for component in sample extract
(area counts or other response units).
VE = final volume of sample extract (mL).
Vx = volume of extract injected onto the HPLC
system (//L) .
13.3 The concentration of aldehyde in the original
sample is calculated from the following equation:
w, mwa
C= - x -xl 0 0 0
A V (orV ) MW,
m s d
where
CA = concentration of aldehyde in the original
sample (ng/L).
Vm or Vs are as specified in Section 13.1.
MWa and MWd are the molecular weights (g/mole)
of the aldehyde and its corresponding DNPH
derivative, respectively.
13.4 The aldehyde concentrations can be converted to
ppbv using the following equation:
CA(ppbv)=CA(ng/L)x-iill
where
CA (ng/L) is calculated using Vs.
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Performance Criteria and Quality Assurance
This section summarizes the quality assurance (QA) measures
and provides guidance concerning performance criteria which
should be achieved within each laboratory.
14.1 Standard Operating Procedures (SOPs)
14.1.1 Each user should generate SOPs describing the
following activities as accomplished in their
laboratory: 1) assembly, calibration and
operation of the sampling system, 2)
preparation, purification, storage and
handling of DNPH reagent and samples, 3)
assembly, calibration and operation of the
HPLC system, and 4) all aspects of data
recording and processing.
14.1.2 SOPs should provide specific stepwise
instructions and should be readily available
to, and understood by, the laboratory
personnel conducting the work.
14.2 HPLC System Performance
14.2.1 The general appearance of the HPLC
chromatograph should be similar to that shown
in Figure 4.
14.2.2 The HPLC system efficiency and peak asymmetry
factor should be determined in the following
manner. A solution of the formaldehyde DNPH
derivative corresponding to at least 20 times
the detection limit should be injected with
the recorder chart sensitivity and speed set
to yield a peak approximately 75% of full
scale and 1 cm wide at half height. The peak
asymmetry factor is determined as shown in
Figure 5, and should be between 0.8 and 1.8.
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14.2.3 HPLC system efficiency is calculated according
to the following equation:
N= 5.54
W,
1/2
where
N = column efficiency, theoretical
plates
tr = retention time of components
(seconds)
W1/2 = width of component peak at half
height (seconds)
A column of efficiency of >5,000 should be obtained.
14.2.4 Precision of response for replicate HPLC
injections should be ± 10% or less, day to
day, for calibration standards. Precision of
retention times should be ± 2%, on a given
day.
14.3 Process Blanks
Prior to use a 10 mL aliquot of each batch of
DNPH reagent should be analyzed as described
in Section 11. In general, formaldehyde
levels equivalent to >5 ng/L in a 60 liter
sample should be achieved and other aldehyde
levels should be <1 ng/L.
At least one field blank should be shipped and
analyzed with each group of samples. The
field blank is treated identically to the
samples except that no air is drawn through
the reagent. The same performance criteria
described in 14.3.1 should be met for process
blanks.
14.4 Method Precision and Accuracy
14.4.1 Analysis of replicate samples indicates a
precision of ± 15-20% relative standard
deviation can be readily achieved. Each
laboratory should collect parallel samples
periodically (at least one for each batch of
samples) to document their precision in
conducting the method.
14.3.1
14.3.2
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14.4.2 Precision for replicate HPLC injections should
be ± 10% or better, day to day, for
calibration standards.
14.4.3 Method accuracy is difficult to assess because
of the difficulty in generating accurate
gaseous standards. Literature results
indicate (1-3) recoveries of 75% or greater
are achieved for a broad range of aldehydes.
Each laboratory should periodically collect
field samples wherein the impinger solution is
spiked with a known quantity of the compound
of interest, prepared as a dilute methanol
solution. Formaldehyde cannot be spiked in
this manner and therefore a solution of the
DNPH derivative should be used for spiking
purposes.
14.4.4 Before initial use of the method each
laboratory should generate triplicate spiked
samples at a minimum of three concentration
levels, bracketing the range of interest for
each compound. Triplicate nonspiked samples
must also be processed. Recoveries of >70 ±
20% and blank levels of <5 ng/L for
formaldehyde and 1 ng/L for the other
compounds (assuming a 60 liter air sample)
should be achieved.
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References
(1) Grosjean, D., Fung, K., and Atkinson, R., "Measurements of
Aldehydes in the Air Environment", Proc. Air Poll. Cont.
Assoc., Paper 80-50.4, 1980.
(2) Grosjean, D. and Fung, K., "Collection Efficiencies of
Cartridges and Micro-Impingers for Sampling of Aldehydes in
Air as 2, 4-Dinitrophenylhydrazones", Anal. Chem. .54, 1221-
1224, 1982.
(3) Grosjean, D., "Formaldehyde and Other Carbonyls in Los Angeles
Ambient Air", Environ. Sci. Technol. 16, 254-262, 1982.
(4) Riggin, R. M., "Technical Assistance Document for Sampling and
Analysis of Toxic Organic Compounds in Ambient Air", EPA-
600/4-83-027. U.S. Environmental Protection Agency, Research
Triangle Park, North Carolina, 1983.
(5) Annual Book of ASTM Standards, Part 11.03, "Atmospheric
Analysis", American Society for Testing and Materials,
Philadelphia, Pennsylvania, 1983.
(6) Berry, D. A., Holdren, M. W., Lyon, T. F., Riggin, R. M., and
Spicer, C. W., "Turbine Engine Exhaust Hydrocarbon Analysis-
Interim Report on Task 1 and 2", Report on Contract No. F-
08635-82-C-0131, Air Force Engineering and Services Center,
Tyndall AFB, Florida, 1983.
(7) Shiner, R., Fuson, R., and Curtin, D., "The Systematic
Identification of Organic Compounds", John Wiley and Sons,
Inc., 5th ed., New York, 1964.
(8) "Method 6 Determination of S02 Emissions from Stationary
Sources", Federal Register, Vol. 42, No. 160, August 1977.
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TABLE 1. ALDEHYDES AND KETONES FOR WHICH THE METHOD HAS BEEN EVALUATED
4444444444444444444444444444444444444444444444444444444444444444444444444444444444444444
Typical
Relative
Molecular Weight Retention
Compound
Derivative
Compound
Time
(a)
Formaldehyde
Acetaldehyde
Acrolein
Propanal
Acetone
Crotonaldehyde
Isobutyraldehyde
Methyl Ethyl Ketone
Benzaldehyde
Pentanal
o-Tolualdehyde
m-Tolualdehyde
p-Tolualdehyde
Hexanal
210
224
236
238
238
250
252
252
286
266
300
300
300
280
30
44
56
58
58
70
72
72
106
86
120
120
120
100
1 . 0
1.3
1 . 6
1.7
1 . 9(b)
2 . 3
2.4
2 . 8
3.2
3.7
4 . 8
5.1
5.3
5.7
4444444444444444444444444444444444444444444444444444444444444444444444444444444444444444
(a) Using HPLC conditions shown in Figure 4.
Formaldehyde = 1.0
(b) Acetone background levels in the reagent prevent its
determination in most cases.
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FIGURE 1. TYPICAL SAMPLING SYSTEM
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SAMPLING DATA SHEET
(One Sample Per Data Sheet)
PROJECT:
SITE:
LOCATION:
INSTRUMENT MODEL NO:
PUMP SERIAL NO:
SAMPLING DATA
DATE(S) SAMPLED:
TIME PERIOD SAMPLED:
OPERATOR:
CALIBRATED BY:
Sample Number:
Start Time: Stop Time:
*Dry Gas* * Flow *Ambient*Barometric* *
* Meter *Rotameter*Rate,*Q* Temp. ^Pressure, *Relative *
Time*Reading* Reading *ml/Min * °C * mmHg *Humidity,%* Comments
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^ * * * * * *
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N * * * * * * *
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Total Volume Data**
Vm = (Final - Initial) Dry Gas Meter Reading, or = Liters
= 0], + + x 1 = Liters
N 1000 x (Sampling Time in Minutes)
Flowrate from rotameter or soap bubble calibrator (specify which).
Use data from dry gas meter if available.
FIGURE 2. EXAMPLE SAMPLING DATA SHEET
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FIGURE 3.
TYPICAL HPLC SYSTEM
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Column - Zorbax ODS, 250 x 4.6 mm
Mobile Phase - 80/20 Methanol/HzO
Flow Rate - 1 mL/Minute
Detector - UV at 370 nm
FIGURE 4.
TYPICAL HPLC CHROMATOGRAM
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FIGURE 5. PEAK ASYMMETRY CALCULATION
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